Method for the urinary detection of bladder cancer

ABSTRACT

The invention relates to the diagnosis of bladder cancer and more specifically to the detection in urine samples of bladder carcinomas of the transitional type. The detection method according to the present invention enables, through the utilization of a DNA chip designed for this purpose, to determine the grade of the detected tumors.

The invention relates to the diagnosis of bladder cancer and moreparticularly to the urinary detection of the transitional type ofbladder carcinoma (TCC: translational cell carcinoma).

Carcinomas of the transitional type are encountered in transitionalepithelium tumors, which comprise approximately 70% to 90% of epithelialbladder tumors. They are characterized by their great morphologicalvariability, so that their prognosis is sometimes difficult to predict.

In terms of histology, urothelial tumors can be papillary ornon-papillary, of high or low malignancy (histological grade),infiltrating or non-infiltrating, each lesion being the combined resultsof these three morphological characteristics.

There are several classifications for urothelial tumors of the bladder.The hitherto most commonly used classification was that of the WHO,which dates back to 1974. It classified transitional cancers of theepithelium as benign (exophytic papilloma, inverted papilloma) ormalignant (transitional epithelial carcinomas of grades 1, 2 and 3). In1998, following several meetings involving anatomic pathologists,urologists, oncologists and biologists, a consensual classification ofurothelial tumors was established and validated. It is this newclassification (Epstein J. I., Am. J. Surg. Pathol. (1998), 22:1435-1448) which is currently used. Although only slightly differentfrom the previous WHO classification, it has the advantage of being incloser agreement with biology and the evolutive potential of urothelialtumors.

Following this classification, bladder tumors are classified by “grade,”from G1 to G3, according to different cytomorphological criteria. Thehigher the grade, the less differentiated is the cells' appearance andthe more aggressive they are.

Tumors are also classified according to their surface characteristics(pTa and T1) or invasiveness in the muscle (from T2 to T4). Low-gradetumors are generally non-invasive or have infiltrated the superficiallayers (stages Ta and T1), whereas high-grade tumors are more aggressiveand are often detected at T1 or a more advanced stage.

The determination of the grade of the tumor cells has a criticalimportance for the clinician, as it will help decide which therapeuticmethods should be used.

Whereas the presence of invasive grade 3 tumors requires total ablationof the bladder and its accessory glands, most low grade/stage tumors (Taand T1) can generally be treated locally with the organ remainingintact, by means of different therapeutical approaches such aschemotherapy, immunotherapy or BCG therapy.

Patients affected by a low-grade, non-invasive tumor generally have agood prognosis, but have to be periodically followed up since the riskof cancer relapse is of the order of 70%. Consequently, the patientsmust be monitored on a regular basis following treatment, every threemonths for the first two years, and every six months thereafter, and inthe case of a relapse it is very important for the tumors' progressionto be monitored.

Moreover, low-grade Ta tumors invade the muscle in only 10-15% of cases,whereas T1 tumors progress to grade T2 in 30-35% of cases. High-gradetumors progress more rapidly, with patients quickly reaching grade T2,and remote metastases often forming during the course of the followingtwo years.

Currently, only invasive clinical examinations, in other words involvingbiopsy, direct endoscopic visualization of the tumors, or surgery canenable a reliable diagnosis to be made of the grade of the tumor cells,and an appropriate therapy to be implemented.

Still, endoscopy is sometimes unreliable and difficult to interpret. Asfor cytology following a biopsy, various studies show that itsreliability is mediocre, resulting in the failed detection of 50% oftumors (Boman, H., et al., 2002, J. Urol., 167(1): 80-83).

On account of the high prevalence of bladder cancer in the population,in particular in persons older than 50, and with the aim to improve boththe diagnosis and the comfort of patients, considerable research effortshave been devoted to the development of non-invasive, indirect detectionof bladder cancer.

In this respect, several research teams have tried to detect urologicaltumors by means of urinary samples using conventional tumor markerproteins.

Nevertheless, until now, the detection of these proteins has not beenfound to be sufficiently specific to be reliable with regard to theorigin of the proteins and the stage of development of the tumors.

In 1999, an American team described the possibility of following theprogression of bladder cancer by analyzing microsatellite DNA sequencescontained in the urine (Steiner et al. (1997) Nature Medicine 3:621-624). This detection method, which consists in amplifying andmeasuring the variability of some repeated, non coding mitochondrial DNAsequences, is based on the observation that these sequences aregenerally altered in carcinoma cells.

Nevertheless, this technique is not entirely satisfactory, insofar as itdoes not effectively enable the grade of the tumors to be determined.

Other molecular methods are based on PCR methods, which attempt toamplify or detect genetic markers, such as local mutations in tumorsuppressing genes, applied to the DNAs present in the patients' urine.However, these methods require specific initiators comprising 20 to 40nucleotides, which are not straightforward to develop, in view of thesize and variability of the human genome. In addition, as soon as theDNA of tumor cells is mixed in the urine with the DNAs of healthy cells,the desired amplification is masked by that caused by the latter.

As a result of the difficulty in employing the different known geneticmarkers of the transitional type of bladder carcinoma, the inventorsprefer the use of an innovative approach based on the simultaneousdetection of several genetic anomalies frequently encountered in TCCcells.

For this purpose, they listed the main genetic anomalies encountered intransitional bladder carcinomas, then selected those most representativeof the grade of the tumor cells. In this approach, they privilegedgenetic anomalies consisting of physical alterations to the chromosomes,such as ploidies or deletions, typically resulting from the analysis ofthe karyotype of tumor cells. They then developed an innovativeprocedure enabling simultaneous detection of the presence or absence ofgenetic anomalies selected in the DNA contained in the patients' urine.

More particularly, the inventors have selected a specific array of 25human chromosome loci (using array-based chromosome hybridizationcomparative analysis techniques with respect to total DNA extracted froma urine sample) providing a significant fluorescent signal.

Until now, tests based on urine samples which offer the same levels ofspecificity and sensitivity, and which are also able to clearlydistinguish between low and high grade tumors have not been madeavailable (Budman L. I. et al., 2008 CUAJ 2(3):212-221).

This method has an astonishing ability to diagnose the presence oftransitional carcinoma cells of the bladder, with no need for surgicalexamination of the bladder, whilst providing a good indication of thegrade of these tumor cells.

FIG. 1: Classification of the tumors analyzed in the examples, accordingto the usual clinical criteria.

FIG. 2: classification of the patients whose urine was analyzed in theexamples, as a function of the results obtained using endoscopy andcytoscopy.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present application is to provide a method for thedetection of bladder cancer, more particularly the urinary detection ofbladder cancer.

This detection is obtained through the marking of the total quantity ofDNA in a patient's urine, and the detection in this DNA, of chromosomesequences comprised in loci, which could potentially be affected bygenetic anomalies.

These chromosome sequences, which are the reference DNAs according tothe present invention, could potentially be lacking in the case ofdeletion of the chromosome locus to which they belong, or duplicated, asfor example in the case of a trisomy.

Tumor cells in bladder cancer are frequently the source of chromosomalreorganizations, indicative to a certain extent of the tumoral characterof the cells. The regions affected by this chromosomal reorganizationare however potentially numerous and of variable extent. In effect,although some loci, in particular those which code for tumor suppressinggenes or are involved in cell-cycle regulation, are altered moresystematically, chromosome anomalies arise when intra andinter-chromosomal recombinations occur between homologous regions, whichare not always the same.

According to a first embodiment, one aim of the present invention is toprovide a method for the in-vitro detection of bladder cancer in apatient, characterized in that it comprises one or more of the followingsteps:

In a first step (A), the DNA contained in a urine sample taken from saidpatient is extracted using one of the numerous methods known to oneskilled in the art. As mentioned above, the present inventionadvantageously allows the cancerous condition of the bladder to bedetermined based on the patients' urine. This method must thereforeconcentrate the DNA contained in the urine sample, which DNA is in itsresidual state since it is derived, in part, from cells present in thebladder. Since the DNA is not a highly fragile material, it is notnecessary to take drastic measures in order to preserve the urinesamples to be analyzed. The samples can thus be taken at the patients'home. The extracted DNA is complete, in the sense that it contains theentire recoverable DNA present in the urine, whether or not itoriginates from tumor cells.

In a following step (B), the DNA extracted in step (i) is fragmented,preferably by sonication, into DNA fragments with a size mostly greaterthan 500 base-pairs (bp), preferably greater than 800 bp, and morepreferably between 1000 and 5000 bp. Alternatively, the DNA can bedigested by means of enzymes such as restriction enzymes.

In a following step (C), the obtained DNA fragments are markeduniformly, by means of a first marking agent so as to form a pool ofmarked DNA. Thus, it is the total DNA extracted from the urine which ismarked. By “uniformly” it is meant that the marking occurs in anon-specific manner such that two different DNA fragments of identicalsize are marked with the same intensity. To obtain such marking, onepreferably makes use of nucleotides coupled to a fluorophore, which areintroduced into the DNA during the course of a replication step. Thisin-vitro enzymatic replication step can advantageously increase thenumber of copies of marked DNA fragments available in the DNA pool. Themarked DNA pool is comprised preferentially of fragments having randomcuts.

In a following step (D), at least one aliquot is formed from the markedDNA pool, said aliquot being capable of forming, according tocircumstances, the entire marked DNA pool. Each aliquot is then broughtinto contact with one, and preferably several, reference DNAs, each ofthese reference DNAs corresponding to a different DNA sequence includedin the locus of a chromosome likely to be affected by a genetic anomaly.Said genetic anomaly is preferably correlated with a specificdevelopment stage of bladder cancer and more particularly with a stagesuch as that mentioned above. The reference DNA can have been chosen,for example, according to data taken from scientific literature or tobiopsy analysis results.

According to a preferred embodiment of the present invention, thereference DNAs are comprised of DNA sequences of human chromosomesbetween 1,000 and 200,000 base pairs (bp) in length, preferably between2,000 and 180,000 bp in length, and still more preferably between 5,000and 160,000 bp in length, such as those that are cloned in BAC(Bacterial Artificial Chromosome) or YAC (Yeast Artificial Chromosome)clones constructed from human genome sequences. Such BAC clones aredescribed in the publicly accessible databases relevant to the humangenome, such as those of the CNBI or the UCSC.

Thus, whereas it has proven difficult to diagnose bladder cancer basedon isolated markers, in particular markers corresponding to shortgenetic sequences enabling genetic amplification tests to be carried out(PCR, TMA, etc.), the detection method according to the presentinvention is based on a detection method using DNA sequences which aregenerally larger than 600 bp.

However, still using hybridization techniques, the present invention maybe carried out by using shorter hybridization probes as reference DNAs,more particularly oligonucleotides of 30 to 100 bp in length, preferablyof 40 to 80 bp, and more preferably of 50 to 70 base pairs. Theseshorter probes have given rise to results equivalent as those obtainedwith the above BAC clones. Even more accurate and sensitive results wereobtained by using several different probes from the same chromosome locior BAC clones. It also provided a more reliable determination of thetumor grade.

According to a preferred embodiment of the invention, the referenceDNAs, preferably under the form of previously defined DNA probes, arefixed on a solid substrate, which may take the form of microarray asdescribed further on, micro beads or solid particles. Such solidsubstrates and their implementation are widely reported in the art.

The inventors have established that it is preferable to use referenceDNAs whose sequence are included within the loci of human chromosomeschosen from: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24,5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p,14q22-qter, 17p, 19 and 22 as referred to in Table 1. Preferably, themethod according to invention involves reference DNAs comprising the DNAsequences included in each of the following loci present in the humanchromosomes: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24,5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p,14q22-qter, 17p, 19 and 22.

According to the present invention, contact between the marked DNAfragments and the reference DNAs is carried out under conditionsenabling specific hybridization, and preferably under stringentconditions such as those described in the experimental part of thepresent application.

In parallel to step (D), a step (E) consists in preparing a control DNApool taken from one or several individuals who do not suffer frombladder cancer. Ideally, this DNA is derived from urinary samples takenfrom several individuals not affected by cancer, chosen randomly fromthe human population. It is also possible to use commercially prepared,human reference DNA. As in the case of DNA extracted from the patient'surine, the control DNA is uniformly marked by means of a second,preferably fluorescent, marker. This marker is preferably different fromthat used to mark the patient's DNA.

As in step (D), an aliquot of marked control DNA is brought into contactwith the selected reference DNAs, which are identical to those used instep (D). This contacting operation can be carried out separately orjointly, under the same hybridization conditions as those used in step(D). When the contacting operation is carried out jointly, this impliesthat the patient's DNA and the control DNA are both contacted,preferably simultaneously, with the same reference DNA.

In a following step (F) the marked DNA fragments, which were notspecifically hybridized to the reference DNAs in steps (D) and (E), areremoved. This step of washing non-hybridized DNAs is carried outaccording to the procedures known to those in the art, without anyparticular difficulty.

In a following step (G) the intensity of the signal produced by themarked fragments that have hybridized to each of the selected referenceDNAs, are determined. The type and intensity of the signal depends onthe type of marker used. When the first and second markers used haveequivalent intensities, as for example in the case of cyanines 3 and 5,the variations observed between the patient's DNA and the control DNAare directly related to the quantity of hybridized DNA fragments locatedat each reference DNA.

In a following step (H), the deviation between the signals recorded fromthe patient's DNA and from the control DNA is determined for eachreference DNA.

According to a preferred embodiment of the present invention, the DNAfragments taken from the patient in step (C) are divided into two pools,with one being marked using a first marker and the second marked using asecond marker. The control DNA fragments from step (D) are also dividedinto two pools, one being marked using the first marker and the secondusing the second marker. With this procedure, a cross-determination ofthe signal deviation can be determined with the first and then with thesecond marker. If the deviations observed between the control DNA andthe patient's DNA are of the same order, no matter which marker is used,this is indicative of significant deviations

In a following step (I), the cancerous stage of the patient is derivedfrom the deviations observed in the previous step. Thus, according tothe present invention, the greater the number of deviations obtained,the more accurate is the detection of bladder cancer. Furthermore, thedeviations provide a qualitative indication of the stage of the tumorsinvolved, in particular when several loci indicating the tumoral gradeare employed simultaneously.

Alternatively, if a locus is considered to be a highly representativemarker of the tumor cells' malignancy, several reference DNAs can beselected from the same locus in order to verify the result derived fromthis locus.

The method according to the present invention thus enables, in step (I),several reference DNAs to be compared and an extended table of thegenetic anomalies encountered to be established. During this step it maythen be found useful to weight the results obtained for each referenceDNA, in order to take into account, for example, for a tumor at a givenstage, the probability of finding a genetic anomaly at the selectedlocus. When several reference DNAs are used to detect an anomaly on thesame locus, it is also possible to weight the deviation obtained for areference DNA, thereby taking its weight relative to the otherrepresented loci into account. This weighting, or use of constants inorder to set correlations between different loci, can be used to designan algorithm enabling a “theoretical stage” to be determined on thebasis of data collected during step (H).

The above method can be summarized as a method for in-vitro detection ofbladder cancer in a patient, characterized in that it comprises thesteps of:

(i) extracting the DNA contained in a urine sample taken from saidpatient;

(ii) fragmenting the DNA extracted in step (i);

(iii) marking the obtained DNA fragments uniformly with a marking agentso as to form a pool of marked DNA;

(iv) forming at least one aliquot from the pool of marked DNA andbringing each aliquot into contact with a set of reference DNAs, saidcontact being carried out under conditions enabling specifichybridization of the marked DNA fragments with said reference DNAs;

said reference DNAs comprising the DNA sequences included in each of thefollowing loci present in the human chromosomes: 1p, 3q, 8q22qter, 20,5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15,16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22;

(v) eliminating the marked DNA fragments which are not specificallyhybridized to the reference DNAs;

(vi) determining the intensity of the signal produced by the markedfragments hybridized to each of the selected reference DNAs;

(vii) determining, for each reference DNA, the deviations between thesignals obtained in comparison with those obtained with a control DNAfrom a healthy patient;

(viii) deriving the patient's cancer stage from said observeddeviations.

The loci, which are preferably covered by the reference DNAs accordingto the present invention, are summarized in Table 1.

The reference DNAs correspond to one or several sequences included inthe corresponding sequences as referenced by the NCBI (start and endpositions on the genome sequence of each chromosome).

TABLE 1 Localization of the reference DNAs Chromosomal Start of thegenome End of the genome localization sequence sequence 1p 20365560113105490 3q 123351123 182149547 8q22-qter 95030146 143909236 20 15145457934415 5p12-p13 1771703 9682916 9p 14010384 37091069 9q 70785194137897571 18q12 33436616 40743026 1q22-q24 154902972 158452461 5p1771703 9682916 6q22 109464089 111511071  7 3863510 158689815 11q1365805698 73155080 12q15 55617597 78745914 13q 25467720 99788952 1525650963 97412048 16 267091 88643457 17q 26415272 77690512 6q25-q27160256061 170536116 7q 62015315 158689815 8p 345303 38886999 10q42335633 135251915 11p 399850 44925329 14q22-qter 72026416 106339461 17p7436433 21191536 19 9998870 54262315 22 20487250 48028036

The characteristics of the relevant loci can be further defined asfollows:

3p: A region having several candidate genes involved in numerous formsof cancer.

4: A region involved more in tumoral progress than in its initiation.Tumor suppressing genes, targeted for other cancerous pathologies, havebeen described in this region.

5p12-p13: A region involved in tumoral aggressiveness, in particularmetastatic spread.

6q: A region involved in high-grade tumors, invading the muscle. Thegene M6P/IGF2R is noted as a genetic indicator in the 6q26-q27 locus.

8p: A region frequently deleted in high-grade invasive tumors(especially 8p21-pter) including the tumor suppressing genes: EXTL3, WRNand PRLTS.

9p or entire 9: A region often affected early. The p16/MTS1/CDKN2A/INK4Aand p15/MTS2/CDKN2B genes are strong kinase inhibitors in theprogression of the cellular cycle. Often affected in the pTas, p16 is ananti-oncogene which depends on Rb, as will be discussed later. INK4A mayact on the control points of p53 in particular.

10q or entire 10: A minimal 10q11-q21 and 10q24-q25 region: involved inmany other cancers (lymphoma, prostate, colon). This area includesnumerous tumor-suppressing genes. 10q deletion could be a factor leadingto an incorrect prognosis.

11p: A region corresponding to tumoral aggressiveness, the firstthreshold in the possible direction of tetraploidy. Deletion couldinfluence the expression of the KAI1 gene (11p11.2), which is ametastasis-suppressing gene. The EXT2, WT1, TSG101 and TSSC5 genes arealso involved in other cancers.

13q14: A region corresponding to RB1 in the advanced grades and stagesof cancer, RB cooperates with P53. Inactivation of RB facilitatestumoral progression by regulating the initiation of phase S. Other genescould be involved, outside the q14 locus, thus explaining the moreextensive deletions generally detected.

14q22-qter: A region which appears to be associated with tumoralprogression. Deletion could be caused by unbalanced translocation. Atleast one tumor-suppressing gene is present at this locus.

16q: A region associated with tumoral progression. CDH1 codes for Ecadherin, so that the deletion results in clinical and biologicalaggressiveness of the tumor cells. Another gene codes for H cadherin,which normally inhibits cellular growth.

17: A deleted or mutated region, which occurs at a later stage, and isassociated with tumoral progression rather than initiation. Deletion ofp53 at 17p13.1 leads to an increase in the number of genetic alterationsdue to a lack of control of the P53 protein. The HIC1 gene at 17p13.1may also be involved.

18q12 or entire 18: A region typically altered in muscle-invading tumors(T2 or more). Smad4/DPC4 and Smad2/MADR2/hMAD2/JU18-1 act on RB and p15by inactivating them.

1q22-q25: An amplified region corresponding to the transition between Taand T1. P73 is often overexpressed, and thus potentially altered byduplication in this area.

3q23-q24-q25-q26-qter: A region already identified as being prone toalteration, even though its involvement has not been proven.

Isochrome 5p (5p gain and associated 5q loss): A region in muscleinvading tumors, altered by the activation of proto-oncogenes.

6p22: An amplified region which can be associated with a tumoralinvasion due to the possible presence of proto-oncogenes.

7: A region including the ERBB1/EGFR gene at 7p14-p21, coding for an EGF(epidermal growth factor) receptor, which plays a role in cellulargrowth. At 7q31 the cmet gene codes for another growth receptor, HGF/SF(hepatocyte growth factor/scatter factor). These areas appear to beinvolved in chaotic cell growth of tumors.

8q: A region often involved in the Ta-T1 transition and the progressiontowards an invasive and especially metastatic tumor, through thepresence of oncogenes in the 8q22-q23 region.

10q22-q23: A region which is rarely amplified, and more commonly deleted(see above).

11: Amplification of at least the 11q13 band with the CCND1 gene whichcontrols entry into the cellular cycle. CCND1 is activated in manycancers but seems to have little correlation with cancer progression.This amplification would entail a relationship with the early cancergrowth stages.

12q15: A region containing the MDM2 gene, which interacts with p53 insome tumors in order to suppress its role by masking its activationsite.

13q33-q34: Possible trisomy 13, found occasionally in bladder tumors,could lead to duplication of this region possibly containing aproto-oncogene.

17q: A commonly duplicated region including the ERBB2 gene at 17q21.This anomaly is more common in the advanced grades and stages, orassociated with relapses.

18q11: A region recognized for some rare significant amplifications.

20: A common region, most often restricted to the 20q locus. Carcinomaproto-oncogenes of the familial form are present at the 20q11-q12 locus.At 20q13, STKIS/BTAK codes for a protein which induces chromosomeinstability.

According to a preferred aspect of the invention, the in-vitro bladdercancer detection method involves testing for ploidy of the DNA of thepatient's urinary cells based on one, several, or all of the followingloci: 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27,7q, 8p, 9p, 9q, 10q, 11p, 14q22-qter, 17p, 18q12, 19 and 22.

According to an alternative embodiment of the present invention, saidmethod involves the testing of DNA ploidy in the patient's urinary cellsfor one or several of the following loci:

-   -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12 and 17p;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p and 11p;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p and 7q;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q and        1q22-24;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24 and 11q13;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13 and 8p;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p and 14q22;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22 and 22;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22 and 5p;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p and 6q22;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22 and 7;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7 and 12q15;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15 and 15;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15 and        6q25-q27;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27        and 19;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27,        19 and 13q;    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27,        19, 13q and 16; and    -   1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q,        1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27,        19, 13q, 16 and 17q.

A further aspect of the present invention involves the use of one ormore reference DNAs taken from the 25 loci indicated above in order todetermine the grade and aggressiveness of a tumor.

Preferably, the number of different reference DNAs useful for theimplementation of the method lies between 2 and 500, preferably between10 and 400, and still more preferably between 50 and 400.

When using reference DNAs under the form of probes, which are generallyof 30 to 100 pb in length, as previously defined, the number of thedifferent probes deposited on the solid support can be much higher,generally from 500 to 100 000, more generally from 1000 to 60 000probes.

In this respect, the present invention also relates to a DNA microarrayor chip, useful for the detection of bladder cancer, more particularlyfor the urinary detection of this cancer, characterized in that itcomprises on its surface several reference DNA deposits, distinct fromone another, each of these deposits corresponding to a sequence includedin one or more of the loci described above. This DNA microarray or chip,which, preferably, is a glass slide, allows the method according to thepresent invention to be implemented over a reduced surface area thusenabling simultaneous comparison of the haploid or polyploid state ofthe patient's DNA in the normal diploid state of a healthy DNA for one,several or all of the above mentioned loci. Such a DNA microarray forthe detection of bladder cancer according to the invention is preferablycharacterized in that it comprises on its surface between 10 and 1000reference DNA deposits, said reference DNAs comprising sequencesincluded in every of the above loci of human chromosomes: 1p, 3q,8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13,12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19and 22. Preferentially, each reference DNA deposit comprises a sequenceincluded in the above loci of the human chromosomes.

In accordance with the method previously described, the DNA microarrayaccording to the invention ideally comprises between 10 and 1000,preferably between 10 and 400 DNA, and more preferably between 300 and400 reference DNA deposits as defined above on its surface. The numberof the deposits is reduced in comparison to genomic microarrays forgenome wide analysis to diminish background noise. This number is deemedoptimal by the inventors to improve the test sensibility with respect tothe detection of bladder cancer, using the BAC clones.

Microarrays involving DNA probes are described in the literature [André,F. et al. (2009) Molecular characterization of breast cancer with highresolution oligonucleotide comparative genomic hybridization array,Clin. Cancer. Res. 15(2):441-451]. The specific microarrays according tothe invention, which are designed to carry out the detection of bladdercancer from urine samples, comprise the probes selected in thechromosome loci as previously detailed.

By DNA chip is meant a flat glass, silicon or plastic substrate, onwhich known nucleic DNA sequences, such as the reference DNAs accordingto the present invention, are placed.

Preferably, according to the present invention, the reference DNA placedon the DNA chip comprises the DNA sequences included only at the loci onwhich the chromosomal anomalies related to bladder cancer have beendescribed, and preferably at one, several or all of the loci describedin the present application.

The present invention also relates to a kit containing one or more ofthe following elements:

-   -   one or more reference DNAs according to the present invention as        described in the foregoing, attached to a glass substrate,        preferably in the form of a DNA chip or microarray;    -   a DNA control sample taken from a healthy patient;    -   a total DNA marker reagent as described in the foregoing; and    -   a software program enabling the comparison between the diploid        state of the patient's DNA and the normal diploid state of a        control DNA to be analyzed.

Such a kit is preferably packed into a box such as to accommodate all ofthe tubes, flasks and reagents needed to implement the method accordingto the present invention.

Further features and advantages of the invention are provided in thefollowing non-limiting examples:

EXAMPLES A—BAC Clones Microarray Preparation and Analysis

I—Preparation of the Glass Slide

Thanks to the availability of public databases (UCSC and NCBI), a seriesof BAC clones (DNA portions from the human genome) were selected tocover the chosen markers listed in Table 1. The position of these cloneswas confirmed by sequencing the two ends of the cloned DNA in order toverify the exact position of the DNA sequence in the genome. Each ofthese clones forms a reference DNA as implied by the present invention.

The totality of selected clones covers all of the chromosomal locichosen as markers, making a total of 341 BAC clones.

The DNA was extracted from the BAC and amplified using repliG technology(Qiagen, Germany). Each DNA was placed so as to form 341 wells in a PCRplate, each containing 10 μg of purified DNA. By means of a Bioroboticrobot and 4 needles, the DNA was placed in very small quantities onto aglass slide. The design, produced by ArrayGenomics, incorporates twohybridization areas enabling, as a precaution, a twin experiment to becarried out. Each DNA is randomly placed 5 times in each hybridizationarea in order to obtain 5 independent measurements.

The DNA was then chemically fixed onto a glass slide enabling it to bepreserved for 3 to 6 months.

II—Genetic Profile of Bladder Tumors

Frozen tumors were collected. The chromosomal DNA of these tumors isextracted using a commercial kit. The tumors considered to be of a “lowgrade” are at stage pTa/G2. The tumors considered to be of a “highgrade” are at least at stage pTa/G3, pT1 and pT2. The tumors areclassified according to the standard clinical criteria (FIG. 2).

The DNA extracted from the frozen tumors is quantified by fluorometry(QuBit system, Invitrogen).

The human genomic DNA used as a control sample for the implementation ofthe diagnostic method is commercial DNA (promega, human genomics DNA).

III—DNA Marking and Hybridization with the Reference DNAs

Marking of the Entire DNAs

-   -   1. 2.3 μg of control DNA is fragmented by sonication in a 55 μl        eppendorf tube using a sonicator (Elmasonic sonicator) for 12 to        13 seconds.    -   2. 200-300 ng of DNA prepared by this means are checked by        electrophoresis on an 0.8% agarose gel, in order to verify that        the DNA is correctly fragmented and that most of the fragments        has a size greater than 600 bp.    -   3. The fragments are purified in micro-columns        (NucleospinExtract II).    -   4. 25 μl (˜1 μg) aliquots of purified DNA and control DNA are        placed in centrifuging tubes for marking with Cy5 and Cy3        fluorophores.    -   5. 20 μl of random sequence primers (tube 1, Enzo Kit) are added        to each tube, then vortexed and centrifuged.    -   6. The tubes are warmed in a water bath at 99° C. for 10        minutes.    -   7. The tubes are placed in ice for 5 minutes then briefly        centrifuged and returned to the ice.    -   8. 5 μl of a mixture of nucleotides marked with cyanin are added        to each tube (tube 2 or 3, Enzo Kit).    -   9. 1 μl of Klenow enzyme fragments (Tube 4, Enzo Kit) is further        added.    -   10. The mixture is incubated in a heat-block for 3 hours and 30        minutes at 37° C.    -   11.5 μl of Stop Buffer (tube 5, Enzo Kit) is added following        which the tubes are placed onto ice.        Washing of the Marked DNA    -   12. 200 μl of NT buffer per 100 μl of DNA are added to the        tubes.    -   13. The contents of each tube are transferred to a column, and        then centrifuged for 1 minute at 11,000 g.

14. 600 μl of a NT3 washing buffer is placed in each column, and thencentrifuged for 1 minute at 11,000 g.

-   -   15. The columns are centrifuged for 2 minutes at 11,000 g.

16. 51 μl of sterile water is placed at the top of the column for 1minute.

-   -   17. The column is centrifuged for 1 minute at 11,000 g and the        dissolved marked DNA is recovered in a tube.        Preparation of the DNA before Hybridization    -   18. 50 μl of Cot-1 DNA (Roche) was added to each tube.    -   19. The volume is adjusted to 200 μl using 0.3M of sodium        acetate (pH 5-8).    -   20. 300 μl of frozen ethanol was added.    -   21. The mixture is vortexed and then incubated in darkness at        70° C. for 15 minutes.    -   22. The tubes are centrifuged for 15 minutes at 11,000 g between        4 and 8° C.    -   23. The supernatant is removed using a vacuum pump.        Preparation of the BAC on a Glass Slide

The DNAs in the form of BAC clones are placed in dehydrated form on aglass plate then fixed using a cross-linkage agent sensitive to UV light(350 mJ).

Hybridization of Marked DNA/Reference DNA (BAC)

-   -   24. The residue recovered in point 23 is added to 5 μl of ddH2O.    -   25. 10 μl for 50 μg/μl of tRNA yeast (Invitrogen, Cat        #15401-011) and the mixture is stirred and warmed to 95° C. for        5 minutes.

26. 15 μl of hybridization buffer (Ambion Slide Hyb Buffer #3) preheatedto 70° C. is added.

-   -   27. The mixture is stirred vigorously for 30 seconds and        centrifuged at 13,000 rpm for 30 seconds.    -   28. The mixture is warmed to 37° C. for 30 minutes.    -   29. The glass slide is entirely covered by the mixture.    -   30. A clean Lifterslip leaf (24×32 inches) (Erie Scientific, Cat        #25X60I-2-4789) is placed above the glass slide in order to        cover the microarray.

31. 20 μl of water was added in order to humidify the seals.

-   -   32. The closed hybridization chamber (Hybridization chamber,        Corning, Cat #2551) is immersed in a water bath at 55° C. for 16        hours.        Post-Hybridization Washing    -   33. The glass plate is incubated in a first solution of 0.1% SDS        0.1×SSC, at 50° C.

The microarray is then placed successively in the following washingsolutions (in each solution, for 45 sec while stirring and for 45 secwithout stirring):

-   -   a) 0.1×SSC, 0.1% SDS;    -   b) 0.1×SSC, 0.1% SDS;    -   c) 0.1×SSC;    -   d) 0.1×SSC;    -   34. The glass slide is immersed at room temperature in a marking        box filled with a 0.1×SSC solution then in another box filled        with 100% ethanol (CML, Cat# BVITRI-PPL25L).    -   35. The glass slides are then placed in conical 50 ml tubes and        then centrifuged for several seconds to dryness.    -   36. The glass slides are preserved in darkness.

The glass slides are analyzed using a fluorescent scanner (GENEPIX4000B) with a sensitivity adjusted according to the intensity of themarking signal.

The results are analyzed using the image analysis software BacMagic+(developed by ARRAYGENOMICS) to graphically visualize the profiles inthe dye swap mode. For each DNA, a text file is exported by BacMagic+and formatted so that it can be used by the seeGH software. Deletionsand gains are annotated based on visualization of the dye swap symmetryin BacMagic+.

Using seeGH, for each group, the annotations are compiled in order toderive an overall image of the detected anomalies and their frequency.In these figures, which are similar to a karyotype table, the marksrepresent anomalies (red=loss, green=gain) and their location. The sizesof the marks represent their detection frequency in the patient pool.

Analyzing FIG. 2 it is possible to create a typical profile of eachtumor group according to its grade and stage, which can then be comparedto typical profiles of tumor groups.

TABLE 2 Gains visualized after analysis of the detected anomalies TOTALPOSITIVE BANDS CLONES MINIMUM pTa G2 pTa G3 T1 T2 1q22-24 4 3 2 2 3q 6 52 1 5p 7 6 2 8q22-ter 3 2 1 2 2 1 11q13 7 6 2 12q21-24 5 4 1 15 8 6 1 1612 10 1 2 17q 11 9 1 18q 10 8 2 20q11-ter 10 8 1 20 14 11 1 2 1

TABLE 3 Deletions visualized after analysis of the detected anomaliesTOTAL POSITIVE BANDS CLONES MINIMUM pTa G2 pTa G3 T1 T2 2q35-ter 2 2 14q26-ter 7 6 1 6q25-27 5 4 1 4 3 8p 14 11 2 2 2 9p 6 5 1 1 9q 12 10 3 22 1 10q 14 11 1 11p 4 3 1 3 11q23-25 5 4 1 1 1 13q13 3 2 2 1 13 7 6 1 12 14q23-31 3 2 2 17p 5 4 1 17 16 13 1 1 18q12 3 2 1 1 18q12-23 11 9 4 194 3 1 1

From this summary, it appears that the pTaG2's are much less alteredthan the higher grades. Some anomalies seem to be characteristic oflow-grade tumors. As described in the literature, deletion 9 is alsofound here, appearing to be more closely related to initiation and oftenassociated with pTas. In the present case, the 3q and 5p gains also seemto be characteristic of the low grades (here, it is not an iso5p asdescribed for higher grades, since there is no 5q gain).

The gain anomalies for the high grades appear gradually: 8q, 1q, 11q butalso 16 and 18, which seem to occur more often in the T2 stages orabove.

The loss anomalies for the high grades are 6q, 11p, and 13, but also18q, the latter event therefore appearing to occur belatedly in thetumor's progression.

IV—Calculation and Definition of Sensitivity and Specificity

Sensitivity: Ability to Detect Sick Patients.

$\frac{{true}\mspace{14mu}{positive}}{{{true}{\mspace{11mu}\;}{positive}} + {{false}\mspace{14mu}{negative}}}$Specificity: Ability of the Test to Determine which Subjects areHealthy.

$\frac{{true}\mspace{14mu}{negative}}{{{true}{\mspace{11mu}\;}{negative}} + {{false}\mspace{14mu}{positive}}}$

Thanks to the use of known tumors by the inventors, it is possible tocompute the test's sensitivity. This will allow the capability of thepresent technique to detect both low-grade and high-grade tumors to beevaluated. Both groups will be separated in the calculations, becauselow-grade tumors are less difficult to detect. Routine tests, such asurinary cytology, detect only 50% of these cancers in their initialphase.

The existing tests are characterized by their sensitivity andspecificity as computed in the scientific literature (expressed by aminimum and a maximum since these values vary according to eachstudy—Sources: Biomarkers for detection and surveillance of bladdercancer, Lorne I. Budman, MSc, Wassim Kassouf, M. D., and Jordan R.Steinberg, MD from the Division of Urology, McGill University HealthCentre, Montreal, Quebec), as indicated in Table 4 below.

TABLE 4 Sensitivity and specificity for the existing tests: TestSensitivity Specificity BTA Stat (Polymedco) 52.5%-78.0% 69.0%-87.1% BTATrak (Polymedco)   51%-100%   73%-92.5% Cytology 12.1%-84.6% 78.0%-100%Hematuria dipstick 47.0%-92.6% 51.0%-84.0% NMP22 Bladder Cancer Test34.6%-100% 60.0%-95.0% (Matritech) NMP22 BladderChek (Matritech)49.5%-65.0% 40.0%-89.8% ImmunoCyt/uCyt+ (DiagnoCure) 63.3%-84.9%62.0%-78.1% ImmunoCyt/uCyt+ and cytology 81.0%-89.3% 61.0%-77.7%UroVysion (Abbott Molecular) 68.6%-100% 65.0%-96.0%

Sensitivity is determined for a given number of markers. For each one ofthese, a sensitivity value can be computed. Obviously, it is notpossible for only one genetic marker to lead to a reliable solution.However, the combination of 25 markers seems to be considerably morejudicious, as shown by the summary assessments in Tables 5 and 6.

TABLE 5 Summary assessment based on the 25 markers Gains Losses Numberof Number of abnormal Low abnormal Locus tumors % Grade Locus tumors %1p 1 10% 5p12p13 1 10% 3q 2 20% 9p 3 30% 8q22-qter 4 20% Total 9q 1 10%sensitivity 80% 11q13 4 29% 18q12 2 20% 19 2 14% 20 2 20%

TABLE 6 Summary assessment based on the 25 markers Gains Losses Numberof Number of abnormal High abnormal Locus tumors % Grade Locus tumors %1q22-24 4 29% 6q25-27 2 14% 3q 3 21% 7q 4 29% 5p 2 14% Total 8p 3 21%sensitivity 86% 6q22 1 14% 9p 1  7% 7 2 14% 9q 3 21% 8q22qter 3 21% 10q1  7% 12q15 1 14% 11p 5 36% 13q 2  7% 13q 4 29% 15 2 14% 14q22qter 3 21%16 1  7% 16 1  7% 17q 1  7% 17p 5 36% 20 1  7% 18q12 4 29% 22 3 21%Specificity: 6 negative DNAs (DNAs without anomalies): 6/(6 + 0))*100 =100%

The 10 anomalies which have been classified in the low-grade group maybe taken as models for tumors in their initiation phase, which have notyet become invasive.

Generally, when the tumor is not invasive but of a high grade, or whenit is already in the T1 or T2 stage or above, the number of anomaliesdetected on the markers of this invention increases.

For the high grades, the method does confirm the importance ofchromosomes 1, 7, 8, 11, 13, 14, 17, and 18.

However, it should be noted that the sensitivity of each of the markersconsidered separately does not exceed 36% (as is the case for cytologyor NMP22 in some studies).

On the other hand, the pooling of all genetic markers selected by theinventors for the present invention provides very good sensitivity, evenfor strictly low grades (the result obtained with the present inventionALONE is as satisfactory as the ImmunoCyt/uCyt+ and cytology combinationused in certain studies).

Similarly, the specificity (computed for normal samples or for DNAsextracted from the urine of patients with negative endoscopy andnegative follow-up results) reaches 100%.

V—Verification on Urine

Urine samples were taken from patients under diagnosis or beingfollowed-up.

Routine endoscopy was performed. The patients were then grouped as shownin FIG. 2.

The method was implemented in the same manner as for the frozen tumors(see part II).

The results are analyzed using the BacMagic+ software to graphicallyvisualize the profiles in the dye swap mode. For each DNA, a text fileis exported by BacMagic+ and formatted so that it can be used by theseeGH software. Deletions and gains are annotated based on visualizationof the dye swap symmetry in BacMagic+.

Using seeGH, for each group, the annotations are compiled in order toderive an overall image of the detected anomalies and their frequency.In these figures, which are similar to a karyotype table, the marksrepresent anomalies (red=loss, green=gain) and their location. The sizesof the marks represent their detection frequency in the patient pool.

For tumors of the higher urinary system, it is possible to derive asummary assessment of the detected anomalies (Tables 7 and 8).

TABLE 7 Summary assessment of the detected anomalies (gains) TOTALPOSITIVE Positive Suspect BANDS CLONES MINIMUM endo endo 1q 10 8 11q22-24 3 2 1  3 19 1 3q 6 5 1 3q26-ter 3 2 1 5p 7 6 1 1 7p 7 6 18q21-23 11 9 2 1 11q13 7 6 1 12 15 12 1 1 12q 7 6 1 13q 7 5.6 1 1 15 86.4 1 16q 5 4 1 1 17p 5 4 1 17q 10 8 4 1 19 4 3 4 1 20 17 14 1 1

TABLE 8 Summary assessment of the detected anomalies (deletions) TOTALPOSITIVE Positive Suspect BANDS CLONES MINIMUM endo endo 1p21-22 4 3 14q12-13 6 5 1 6q25-27 3 2 2 1 8p 14 11 2 1 9p 6 5 5 1 9q 12 10 4 1 10q223 2 1 11p 10 8 1 1 14q23-31 4 3 1 17p 5 4 1 1 17q 10 8 1 18q12 3 2 118q12-23 11 9 5 20q 10 8 1 22q 8 6 1

For patients with positive endoscopy, the anomalies characteristic ofbladder cancers are recognized, with some variations which are listedhere.

Moreover, the anomaly distribution table will be used in an endeavor todetermine the grade of these detected tumors.

For bladder tumors, this is also carried out by deriving a summaryassessment of the detected alterations.

TABLE 9 Summary assessment of the detected anomalies (gains) Suspectpositive TOTAL POSITIVE Positive endo BANDS CLONES MINIMUM endo(follow-up) 1q22-24 4 2 1 3p24-ter 4 3 1 3q 6 5 5p 7 6 2 8q22-ter 3 2 31 10p 4 3 1 11q13 7 6 4 2 12q21-24 5 4 15 8 6 16 12 10 1 17p 5 4 1 17q11 9 2 1 18q 10 8 2 20q11-ter 10 8 3 1 20 14 11 1 1

TABLE 10 Summary assessment of detected anomalies (deletions) Suspectpositive TOTAL POSITIVE Positive endo BANDS CLONES MINIMUM endo(follow-up) 2q35-ter 2 2 4p 3 2 1 4q26-ter 7 6 6q15-24 5 4 1 6q25-27 5 41 1 8p 14 11 2 2 9p 6 5 3 1 9q 12 10 1 1 10q 14 11 1 11p 4 3 1 111q23-25 5 4 1 13q13 3 2 13 7 6 14q23-31 3 2 17p 5 4 17 16 13 18q12 3 22 1 18q12-23 11 9 1 19 4 3

The results listed in Tables 9 and 10 show that many characteristicanomalies are detected in patients with positive endoscopy. However,suspect endoscopies reveal fewer alterations. In patients for whom theendoscopy diagnosis was not fully determined, the follow-up showed thatthese patients had progressed unfavorably. It thus seems that a suspectendoscopy would have been clarified by the inventors' test beforereaching the 6-month follow-up.

It should also be noted that patients with negative endoscopy as well asthose for whom the 12-month follow-up still revealed nothing, showed nopositivity for the tested areas. (Only a few clones showed variations,but these were not areas intended to be tested, and corresponded topolymorphic variations of clones spotted on the slide. These clones willhave to be suppressed in the forthcoming versions of the present slide,because of their varying nature and near-centromeric position.)

If the analysis is performed on a case by case basis on each of the DNAsextracted from the urine of a patient urine with positive endoscopy, thepresent invention's summary table, which combines a low grade or a highgrade, may be applied.

SPECIFIC EXAMPLES

DNA 34016: UUTT with positive endoscopy. The 9p and 9q deletions,indicative of low grades, were detected.

The inventors' urologist partner carried out a biopsy following thetest. A grade 1 or 2 was determined.

DNA 33276: UUTT with positive endoscopy. Numerous anomalies (8 in total)are detected, among which 9q, 11p, 8p and 1q22-q2, which arecharacteristic of high grades. Following the biopsy, a grade G3 wasconfirmed.

DNA 32479: Bladder tumor with suspect endoscopy but positive follow-upat 6 months: a gain of 19 only was detected. This small number ofanomalies and the undetermined influence of 19's gain, allow theinventors to associate this tumor with a low grade. The biopsy indicatedgrade G1/G2.

A biopsy was carried out on most of the patients whose urine was testedin this study, whenever their endoscopy was positive. At the time whenthe BCA test was carried out, the results of the biopsy were notavailable.

This test was thus carried out in the blind mode. Its results can besummarized for bladder cancer as shown in Table 11 below.

TABLE 11 Bladder tumor: positive endoscopy: 8 urine samples BCA BIOPSYDNA # POSITIVE MARKERS CONCLUSION RESULT Fb48 None Low Grade G1 Nonanalyzable 30869 8q22-qter, 11q13, 17q Low Grade G2 Non analyzable 3247919 Low Grade G1 G1/G2 32759 9p Low Grade G1 G1/G2 Fb43 19 Low Grade G1G1/G2 30656 6q25-q27 Low Grade Non analyzable 32671 9p, 6q25-q27 LowGrade G3 progression 31138 None Low Grade Non analyzable 33442 6q27, 8p,17q High Grade G3 32924 5p, 8q, 11q13, 17q, 20, 8p, 9p, Very high G3 9q,10q, 11p, 16, 18q12, 22 grade Fb13 6q25-q27, 9p, 14q22-qter, High GradeG3 11q13, 20, 22 39373 5p, 8q22-qter, 18q12, 20 High Grade G3

It should be noted that for all patients for whom the result of thebiopsy was unambiguous, it would have been possible to predict the gradeof the tumor. Moreover, in some cases, a more accurate analysis of thealtered markers provides information about the aggressive character ofthe tumor, its metastatic potential and its possible progression.Moreover, some biopsies that were too restricted could not be analyzed,whereas the present invention is able to provide a clear resultconcerning the grade of these tumors.

B—Oligonucleotide Microarray Preparation

Similar results as those presented above were obtained by usingmicroarrays in which the reference DNAs are present in the form ofoligonucleotide probes. The protocol carried out in this respect was asfollows:

I—Determination of Integrity and Amount of Genomic DNA (gDNA)

-   -   1. Measure gDNA concentration on Nanodrop ND-1000        spectrophotometer (A₂₆₀/A₂₈₀ ratio should be in range of        1.8-2.0, A₂₆₀/A₂₃₀ ratio should be >2.0),    -   2. Confirm gDNA size by running ˜200 ng of DNA on a 0.8-1%        agarose gel (DNA should be of intact genomic size without any        signs of degradation),    -   3. Sonicate the samples,    -   4. Aliquot 200 ng of sonicated test gDNA and 200 ng of sonicated        reference gDNA into two separate 1.5 ml microcentrifuge tubes.        II—Fluorescent Labeling of Sonicated gDNA    -   1. Thaw and add 12.2 ul Alexa Fluor® 2× Reaction Mixes (from the        BioPrime Total Labeling Module) to the tubes containing        sonicated gDNA    -   2. Incubate samples (while protected from light) at 95° C. for 5        minutes.    -   3. Immediately place the samples on ice (snap cool) for 5        minutes.    -   4. Add 1.5 μl of Exo-Klenow Fragment (from the BioPrime Total        Labeling Module) to each tube (50 ul total volume).    -   5. Mix samples    -   6. Incubate samples (while protected from the light) at 37° C.        for 2 hours.        III—Purification of Labeled DNA    -   1. Add 200 μl of Binding Buffer B2 (from the BioPrime Total        Labeling Module) to each tube and vortex to mix.    -   2. Load each sample containing B2 from Step 1 above onto a        PureLink™ Spin Column (from the BioPrime Total Labeling Module),        pre-inserted into a collection tube.    -   3. Centrifuge at 10,000×g for 1 minute; discard the flow-through        and place the column back in the collection tube.    -   4. Add 650 μl of Wash Buffer W1 (from the BioPrime Total        Labeling Module) to the column.    -   5. Centrifuge at 10,000×g for 1 minute; discard the flow-through        and place the column back in the collection tube.    -   6. Spin at maximum speed for an additional 2-3 minutes to remove        any residual wash buffer; discard the flow-through and the        collection tube.    -   7. Place the spin column in a new, sterile 1.5 ml        microcentrifuge tube.    -   8. Add 12.5 ul of water to the center of the column and incubate        at room temperature for 1 minute.    -   9. Centrifuge at maximum speed for 2 minutes; flow-through        contains the purified labeled DNA probes (discard the column        after use).    -   10. Place 1.5 ul of each sample on the NanoDrop        spectrophotometer to determine its yield and specific activity        (use Microarray option).    -   11. Combine test Alexa Fluor®-3 labeled sample with reference        Alexa Fluor®-5 labeled sample.    -   12. Aliquot 16.0 ul of combined mixture into a new 1.5 ml        microcentrifuge tube.        IV—Preparation of Labeled gDNA for Hybridization    -   1. Add components below in the order indicated in a        nuclease-free tube:        -   16.0 μl AF₅ and AF₃ labeled gDNA mixture;        -   2.0 μl Cot-1 DNA (1 mg/ml)(prepared at 1000 ng/μl            concentration);        -   4.5 μl Agilent 10× Blocking Agent; and        -   22.5 μl Agilent 2× Hybridization Buffer (q.s. 45 μl)    -   2. Mix samples    -   3. Incubate samples (while protecting from light) at 95° C. for        3 minutes.    -   4. Immediately incubate tubes at 37° C. for 30 minutes.    -   5. Spin tubes at maximum speed to collect the sample in the        bottom of the tube.        V—Microarray Hybridization    -   1. Load a clean 8×60K gasket slide into the Agilent SureHyb        chamber base with the gasket label facing up and aligned with        the rectangular section of the chamber base.    -   2. Slowly dispense 45 ul of hybridization sample mixture into        each gasket well in a drag and dispense manner.    -   3. Place an 8×60K microarray slide (“active side” down) onto the        gasket slide.    -   4. Place the assembled slide chamber in the rotator rack in the        rotating hybridization oven set to 65° C.    -   5. Rotate at the samples at 20 rpm for 12-16 hours.        VI—Microarray Washing and Staining    -   1. Remove one hybridization chamber from incubator.    -   2. Disassemble the slide chamber.    -   3. Remove the microarray slide and place into slide rack in the        slide staining dish #2 containing Oligo aCGH Wash Buffer 1 at        room temperature. Minimize exposure of the slide to the air.        Touch only barcode portion of the microarray slide or its edges.    -   4. When all slides are in the slide rack #2, stir (adjust the        setting to get good but not vigorous mixing) for 5 minutes.    -   5. Transfer slide rack to slide staining dish #3 containing        Oligo aCGH

Wash Buffer 2 at 37° C., and stir (adjust the setting to get good butnot vigorous mixing) for 1 minute.

-   -   6. Slowly remove slide rack trying to minimize droplets on the        slides. It should take 5 to 10 seconds to remove the slide rack.        If there are any remaining droplets on the array (DNA surface),        spin the slide in the slide spinner for ˜10 seconds.

The slides were scanned immediately, prior to computer image analysis.

The invention claimed is:
 1. A method for in-vitro detection of bladdercancer, and deriving the stage of the cancer in a patient, comprising:(i) extracting DNA contained in a urine sample taken from said patient;(ii) fragmenting the DNA extracted In step (I); (iii) marking theobtained DNA fragments uniformly with a marking agent so as to form apool of marked DNA; (iv) forming at least one aliquot from the pool ofmarked DNA and bringing each aliquot into contact with a set ofreference DNAs, said contact being carried out under conditions enablingspecific hybridization of the marked DNA fragments with said referenceDNAs; wherein the set of reference DNAs consists essentially ofsequences included in each of the following loci of the humanchromosome: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p,6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p,14q22-qter, 17p, 19 and 22, and wherein the reference DNAs are at least30 bases in length; (v) eliminating the marked DNA fragments which arenot specifically hybridized to the reference DNAs; (vi) determining theintensity of the signal produced by the marked fragments hybridized toeach of the selected reference DNAs; (vii) determining, for eachreference DNA, the deviations between the signals obtained In comparisonwith those obtained with a control DNA from a healthy patient; and(viii) deriving the patient's cancer stage from said observeddeviations.
 2. A method according to claim 1, wherein the reference DNAsare DNA probes of 30 to 100 bases.
 3. A method according to claim 2,wherein said DNA probes are fixed on a microarray, on micro beads or onsolid particles.
 4. A method according to claim 1; wherein saidreference DNAs comprise several sequences included in each of thefollowing loci of the human chromosome: 1p, 3q, 8q22qter, 20, 5p12-p13,9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, Hq13, 12q15, 13q, 15, 16, 17q,6q2S-q27, 7q, 8p, 10q, 11p, 14q22-qter, I7p, 19 and
 22. 5. A methodaccording to claim 1, wherein the reference DNAs are 1,000 to 200,000bases in length.
 6. A method according to claim 5, wherein the referenceDNAs consist of BAC clones of the human genome.
 7. A method according toclaim 1, wherein a first and a second marker are used respectively formarking the urine DNA extracted fragments and the control DNA fromhealthy patient.
 8. A method according to claim 7, wherein the first andsecond markers are fluorescent markers emitting at differentwavelengths.
 9. A method according to claim 1, wherein the grade of thetumor cells in step (viii) is determined as a function of the deviationsobserved with respect to the reference DNAs.
 10. A method according toclaim 1, wherein the reference DNAs are deposited separately on amicroarray, prior to hybridization with the marked DNA fragments.
 11. Amethod according to claim 1, wherein the DNA fragments taken from thepatient in step (iii) are divided into two pools, one being marked witha first marker and the second with a second marker, the control DNA ofstep (vii) being also divided into two pools, one being marked with thefirst marker and the second with the second marker, across-determination of the signal deviation being carried out in step(vii) between each of the thus marked pools.
 12. A DNA microarray forthe detection of bladder cancer comprising, fixed on its surface,deposits of 10 to 1000 reference DNAs; said reference DNAs havingsequences included in each of the following loci of the humanchromosome: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p,6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p,14q22-qter, 17q, 19 and 22, and wherein the reference DNAs are at least30 bases in length.
 13. A DNA microarray according to claim 12 for usein the in-vitro urinary detection of bladder cancer.
 14. A DNAmicroarray according to claim 12, wherein it comprises 10 to 400 of saidreference DNA deposits on its surface.
 15. A DNA microarray according toclaim 12, wherein each of the reference DNA deposits comprises asequence of at least 30 bases included in the following loci of thehuman chromosome: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12,1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p,10q, 11p, 14q22-qter, 17p, 19 and
 22. 16. A DNA microarray according toclaim 12, wherein each of the reference DNA deposits consistsessentially of a sequence of at least 30 bases included in the followingloci of the human chromosome: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q,18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27,7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and
 22. 17. A DNA microarraycomprising, fixed on its surface, reference DNA deposits consistingessentially of DNA sequences of the following loci of the humanchromosome: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p,6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p,14q22-qter, 17p, 19 and 22; and wherein the DNA sequences are at least30 bases in length.
 18. The DNA microarray of claim 17, wherein each ofthe reference DNA deposits consists essentially of DNA sequences fromeach of the loci.
 19. The DNA microarray of claim 17, wherein eachreference DNA deposit is homogeneous to DNA sequences from a specificlocus.
 20. The DNA microarray of claim 17, wherein the DNA sequences are30 to 100 bases in length.
 21. The DNA microarray of claim 17, whereinthe DNA sequences are 1,000 to 200,000 bases in length.
 22. The DNAmicroarray of claim 17, comprising 10 to 1000 references DNA deposits.23. The DNA microarray of claim 17, comprising between 1,000 and 60,000reference DNA deposits.
 24. A method according to claim 5, wherein thereference DNAs are 2,000 to 180,000 bases in length.
 25. A methodaccording to claim 5, wherein the reference DNAs are 5,000 to 160,000bases In length.
 26. A method according to claim 8, wherein the firstand second markers are the fluorescent markers Cy3 and Cy5.
 27. A DNAmicroarray according to claim 12, comprising 300 to 400 of saidreference DNA deposits on its surface.
 28. The DNA microarray of claim17, wherein the DNA sequences are 40 to 80 bases in length.
 29. The DNAmicroarray of claim 17, wherein the DNA sequences are 50 to 70 bases inlength.
 30. The DNA microarray of claim 17, wherein the DNA sequencesare 2,000 to 180/000 bases in length.
 31. The DNA microarray of claim17, wherein the DNA sequences are 5,000 to 160,000 bases in length. 32.The DNA microarray of claim 17, comprising 10 to 400 reference DNAdeposits on its surface.
 33. The DNA microarray of claim 17, comprising300 to 400 of reference DNA deposits on its surface.